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Correlative in vivo 2 photon and focused ion beam scanning electron microscopy of cortical neurons - PubMed

Correlative in vivo 2 photon and focused ion beam scanning electron microscopy of cortical neurons

Bohumil Maco et al. PLoS One. 2013.

Abstract

Correlating in vivo imaging of neurons and their synaptic connections with electron microscopy combines dynamic and ultrastructural information. Here we describe a semi-automated technique whereby volumes of brain tissue containing axons and dendrites, previously studied in vivo, are subsequently imaged in three dimensions with focused ion beam scanning electron microcopy. These neurites are then identified and reconstructed automatically from the image series using the latest segmentation algorithms. The fast and reliable imaging and reconstruction technique avoids any specific labeling to identify the features of interest in the electron microscope, and optimises their preservation and staining for 3D analysis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. In vivo imaging, laser branding and tissue preparation.

A, Cortical surface showing the vasculature on the surface of the brain. Dotted lines indicate the blood vessels that can also be seen as dark shadows in the 2PLSM (B), with the white square indicating the region imaged at higher magnification (inset). After fixation and sectioning this region (C) was then laser branded, and reimaged using 2PLSM. These branding marks were visible (D) in the resin block (indicated with white arrow heads) without any further enhancement. Their position is also highlighted with laser etching on the surface (black arrows) that can be seen in the FIBSEM (E). This indicates the region to be imaged (F) so that imaging and milling will capture the branded region (white arrow heads). Scale bar in A and B is 100 µm, and 10 µm in (C–F).

Figure 2
Figure 2. Interactive segmentation of neurites.

A, The ilastik software allows users to select regions for segmentation (yellow box), and compare the generated 3D model (B) with the in vivo image (C) of the neurite of interest. A GFP dendrite from a layer 5 pyramidal neuron is shown in C. The method can also be used with other fluorescent markers, such as tdTomato (D), here expressed in GABAergic axons and dendrites in a different animal. When the density of neurites (E) is high the neurites can still be distinguished. Panel E, shows the reconstructed axons (red) and dendrites (grey) that are shown in the in vivo image in D. Scale bar in C and D is 5 µm.

Figure 3
Figure 3. Manual reconstruction of a bouton its synaptic partner, and all membrane organelles contained.

A, B, The FIBSEM image series can be used to segment in vivo imaged structures (A, inset) including all their organelles: axonal bouton – yellow, mitochondria – green, synapse – red, synaptic vesicles – gold, endoplasmic reticulum – blue, dendritic spine – pink, dendritic endoplasmic reticulum – orange. The reconstruction is made from an image volume (6.4 µm×8.0 µm×5.0 µm) (B, inset left) that also includes the synaptically coupled dendritic spine (B, inset right). Scale bar in A is 1 µm, and in A (inset) is 5 µm.

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References

    1. Soto GE, Young SJ, Martone ME, Deerinck TJ, Lamont S, et al. (1994) Serial section electron tomography: a method for three-dimensional reconstruction of large structures. Neuroimage 1: 230–243. - PubMed
    1. Chen X, Nelson CD, Li X, Winters CA, Azzam R, et al. (2011) PSD-95 is required to sustain the molecular organization of the postsynaptic density. J Neurosci 31: 6329–6338. - PMC - PubMed
    1. da Costa NM, Martin KA (2009) The proportion of synapses formed by the axons of the lateral geniculate nucleus in layer 4 of area 17 of the cat. J Comp Neurol 516: 264–276. - PubMed
    1. Bock DD, Lee WC, Kerlin AM, Andermann ML, Hood G, et al. (2011) Network anatomy and in vivo physiology of visual cortical neurons. Nature 471: 177–182. - PMC - PubMed
    1. Tapia JC, Kasthuri N, Hayworth KJ, Schalek R, Lichtman JW, et al. (2012) High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy. Nat Protoc 7: 193–206. - PMC - PubMed

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Grants and funding

This work was supported by Swiss National Science Foundation Synergia grants CRF II313470/1 (GK), CRSI33-127289 (AH), 3100AO-120685 (AH), 31003A-135631 (AH), and the IRP/Hans Wilsdorf Foundations (AH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.